Tollip interactions.

<p>A) GST-Tollip interaction with ³⁵S-methionine labelled proteins isolated by the yeast two hybrid technique. Lane 1 contains 1/10 input of the ³⁵S-methionine protein used for each interaction; lanes 2 and 3 contain the elution product from incubation of the ³⁵S-proteins with the GST-Tollip and GST-protein alone, respectively.³⁵S-methionine labelled proteins were visualized by autoradiography. Western blot analysis of 293T cells transfected with Ubc9 and HA-Tollip (B), Flag-ARIP3 and HA-Tollip (D), HA-Cystatin B and Ubc9 (C), HA-Cystatin B and Flag-ARIP3 (E). HA-Cystatin B is a ubiquitous protein with antiprotease function, unrelated to Tollip, nor to the inflammatory pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004404#pone.0004404-DiGiaimo1" target="_blank">[28]</a>. Lane 1 contains the protein extract; lane 2 contains the proteins immunoprecipitated with anti-HA abs. In this and in the following figures, “pe.” refers to the protein extract and “ip.” to the immunoprecipitated protein. Staining carried out as indicated under the figures.</p

Sumoylation of Tollip.

<p>A) Western blot analysis of protein extracts from 293T cells transfected as indicated above the figure and stained for the proteins indicated on the right. B) Western blot analysis of protein extracts from 293T cells transfected with HA-Tollip and SUMO-1 (lane 1) and SUMO-1 only (lane 2) stained with anti-SUMO-1 abs. The same protein extracts were immunoprecipitated with anti-HA abs and stained with anti-SUMO-1 abs (lanes 3 and 4) and, after stripping of the membrane, they were stained with anti-HA abs (lanes 5 and 6). These samples were electrophoresed in 8% SDS-PAGE. C) Western blot of protein extracts from 293T cells transfected and stained as indicated.</p

Cellular localization of Tollip.

<p>A–B) HeLa cells transiently expressing HA-Tollip and Flag-SUMO-1 stained with anti−ΗΑ (Cy5-red) and anti-Flag (FITC-green) abs. E–F) SAOS-2/IL-1RI cells transfected with HA-Tollip only and stained with anti−HA (Cy5 -red) and anti-SUMO-1 abs (FITC- green). I–J) SAOS-2/IL-1RI cells transfected with HA-Tollip only and stained with anti-HA (Cy5 -red) and anti-Daxx (FITC- green) abs. C,D,G,H,K,L) Merged reconstructed images of extended focus projections are shown. The selected areas (white rectangles) are reconstructed as three-dimensional imaging using the surface-shaded algorithm operating above a defined threshold of fluorescence intensity. The detail allows a precise evaluation of localization of the two fluorescent signals. Magnification bar: 5 µm.</p

Tollip and ARIP3 interacting domains.

<p>A) Schematic representation of HA-Tollip deletion mutants. The black box represents the C2 domain (aa 54–179), the grey box represents the CUE domain (aa 229–274). HA-Tollip wt: aa 1–274; Δ1: aa 1–229; Δ2; aa 1–179: Δ3: aa 54–274; Δ4: aa 94–274; Δ5: aa 134–274; Δ6: aa 179–274; Δ7: aa 54–179. In all western blots described in this figure, Lane 1 contains the protein extract and lane 2 the immunoprecipitated proteins. B) Western blot of protein extracts from 293T cells, transfected with Flag-ARIP3 cDNA and with the indicated HA-Tollip deletion mutants, before (lane 1) and after (lane 2) immunoprecipitation with anti-HA abs. The staining is with anti-Flag abs. C) Western blot from the same protein extracts as in B stained with anti-HA abs. D) Schematic representation of the Flag-ARIP3 deletion mutants. The circle represents the SAP domain (aa 11–45), the white box with black stripes represents the RING domain (aa 347–388) and the black box with white stripes represents the AR-ID domain (aa 443–548). Flag-ARIP3 wt: aa 1–572; Δa: aa 1–467; Δb: aa 1–347; Δc: aa 1–169. E) Western blot of protein extracts from 293T cells transfected with HA-Tollip and the indicated Flag-ARIP3 deletion mutants. The protein extract was immunoprecipitated with anti-HA abs and stained with anti-Flag antibodies. F) Western blot of protein extracts from 293T cells transfected with the Flag-PIAS-1 cDNA together with HA-Tollip wt and mutants as indicated. Immunoprecipitation with anti-HA abs; staining with anti-Flag abs. G) Western blot of protein extracts from 293T cells transfected with the Flag-PIASxβ cDNA together with the HA-Tollip wt and mutants as indicated. The protein extract was immunoprecipitated with anti-HA abs and stained with anti-Flag abs. H, I) Western blots of the same samples as in F and G respectively, stained with anti–HA abs.</p